The interstitial matrix is structurally diverse connective tissue found in the dermal and stromal layer of skin and cornea, tendon, cartilage, blood vessel walls and around muscle fibers. It consists of a highly ordered structure of collagen fibrils, glycoprotein and proteoglycans. Interactions between these macromolecules provide the architectural framework for the various interstitial matrices. The proteoglycans of the interstitial matrix contain structurally related core proteins that are, however, products of distinct genes and are collectively termed the small interstitial proteoglycans (SIPG). Lumican is a keratan sulfate SIPG with wide spread occurrence in connective tissues of dermis, corneal stroma, arterial walls and muscle. The deduced primary structure of the chicken, bovine and human core protein indicate the presence of three structural domains. Domains I and III contain 4 and 2 cysteine residues. The largest central domain contains multiple beta sheet forming leucine repeats that are likely to be involved in much of its interactions with other proteins. In vitro lumican binds collagen and inhibits collagen fibrillogenesis. In vivo it is believed to play a fundamental role in collagen fibril structure and organization. The long term goals of this study are to use genetic approaches to elucidate lumican's functions in interstitial matrices. Lumican deficient mice will be generated using gene targeting techniques to determine the essential function of this proteoglycan in connective tissue organization. The first aim of this project is to develop a targeting vector, introduce it into embryonic stem (ES) cells and isolate lum+/lum-ES cells which will be used for generating lumican deficient mouse. The second aim is to isolate mouse lumican cDNA clones, determine its nucleotide sequence and use this to make lumican antisense RNA probes for expression studies. The third aim is to express the cDNA as a recombinant protein and use this and an N- terminal synthetic peptide to generate antibodies against mouse lumican to localize the core protein in tissues. The antisense RNA and antibody probes will allow close monitoring of lumican expression in cells and its accumulation in interstitial matrices during development and in normal adult mice. These findings will form the basis for understanding phenotypic abnormalities associated with lumican deficiency. The experiments described in these aims will provide the framework for new insights into lumican's functions in connective tissues and will lead to animal models for various connective tissue disorders.